Enhanced lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. dhg

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of E. coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

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Enhanced Lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. Dhg

Catalysts ◽

10.3390/catal9121003 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1003 ◽

Cited By ~ 1

Author(s):

Min Jae Kim ◽

Myung Hyun Noh ◽

Sunghwa Woo ◽

Hyun Gyu Lim ◽

Gyoo Yeol Jung

Keyword(s):

Escherichia Coli ◽

Cell Membrane ◽

Electron Transport Chain ◽

Farnesyl Diphosphate ◽

Mep Pathway ◽

Farnesyl Diphosphate Synthase ◽

Mva Pathway ◽

Transport Chain ◽

Lycopene Production ◽

Specific Activities

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of Escherichia coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

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Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01402-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7291-7293 ◽

Cited By ~ 44

Author(s):

Gopal Prasad Ghimire ◽

Hei Chan Lee ◽

Jae Kyung Sohng

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Streptomyces Peucetius ◽

E Coli ◽

Expression Of Genes ◽

Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

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The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K 12

Biochemical Journal ◽

10.1042/bj1250489 ◽

1971 ◽

Vol 125 (2) ◽

pp. 489-493 ◽

Cited By ~ 32

Author(s):

G. B. Cox ◽

N. A. Newton ◽

J. D. Butlin ◽

F. Gibson

Keyword(s):

Escherichia Coli ◽

Energy Source ◽

Electron Transport ◽

Electron Transport Chain ◽

Adenosine Triphosphatase ◽

E Coli ◽

Transport Chain ◽

Mutant Strains ◽

K 12 ◽

A Site

1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg2++Ca2+-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.

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Metabolic Engineering Escherichia coli for the Production of Lycopene

Molecules ◽

10.3390/molecules25143136 ◽

2020 ◽

Vol 25 (14) ◽

pp. 3136 ◽

Cited By ~ 2

Author(s):

Zhaobao Wang ◽

JingXin Sun ◽

Qun Yang ◽

Jianming Yang

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Regulatory Networks ◽

Carbon Sources ◽

Operation Technique ◽

Mva Pathway ◽

E Coli ◽

Microbial Cell Factories ◽

Cell Factories ◽

Lycopene Production

Lycopene, a potent antioxidant, has been widely used in the fields of pharmaceuticals, nutraceuticals, and cosmetics. However, the production of lycopene extracted from natural sources is far from meeting the demand. Consequently, synthetic biology and metabolic engineering have been employed to develop microbial cell factories for lycopene production. Due to the advantages of rapid growth, complete genetic background, and a reliable genetic operation technique, Escherichia coli has become the preferred host cell for microbial biochemicals production. In this review, the recent advances in biological lycopene production using engineered E. coli strains are summarized: First, modification of the endogenous MEP pathway and introduction of the heterogeneous MVA pathway for lycopene production are outlined. Second, the common challenges and strategies for lycopene biosynthesis are also presented, such as the optimization of other metabolic pathways, modulation of regulatory networks, and optimization of auxiliary carbon sources and the fermentation process. Finally, the future prospects for the improvement of lycopene biosynthesis are also discussed.

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Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Synthetic Biology ◽

10.1093/synbio/ysz003 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 36

Author(s):

Quentin M Dudley ◽

Connor J Nash ◽

Michael C Jewett

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Enzymatic Synthesis ◽

Biosynthetic Pathway ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Design Criteria ◽

Acetyl Coa ◽

E Coli ◽

Enzymatic Pathways

Abstract Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l−1) limonene over 24 h, a productivity of 3.8 mg l−1 h−1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

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Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli

Natural Product Communications ◽

10.1177/1934578x1601100905 ◽

2016 ◽

Vol 11 (9) ◽

pp. 1934578X1601100 ◽

Cited By ~ 1

Author(s):

Takahiro Kato ◽

Jung-Bum Lee ◽

Futoshi Taura ◽

Fumiya Kurosaki

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Immunoblot Analysis ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Transformed Cells ◽

Bacterial Cells ◽

E Coli ◽

Enhanced Production ◽

Escherichia Coli Cells

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and farnesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 μg/mL culture), however, the concentration of the compound elevated to 0.08 μg/mL culture in the cells transformed by GS plus FPS δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 μg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient 6-guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

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Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea

Applied and Environmental Microbiology ◽

10.1128/aem.00950-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 5

Author(s):

Nikolas Duszenko ◽

Nicole R. Buan

Keyword(s):

Escherichia Coli ◽

Electron Transport ◽

Stationary Phase ◽

Exponential Growth ◽

Electron Transport Chain ◽

Methanosarcina Barkeri ◽

Metabolic Efficiency ◽

Methanosarcina Acetivorans ◽

Content Type ◽

Transport Chain

ABSTRACT Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh. IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.

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Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.180.7.1814-1821.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1814-1821 ◽

Cited By ~ 52

Author(s):

Yong Yang ◽

Ho-Ching Tiffany Tsui ◽

Tsz-Kwong Man ◽

Malcolm E. Winkler

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Specific Activity ◽

Salvage Pathway ◽

Pyridoxal Kinase ◽

Triple Mutant ◽

Reading Frame ◽

E Coli ◽

Specific Activities ◽

K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

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